Glucose transport and metabolism are critical for mammalian blastocyst formation and further development. We have previously shown that any decrease in glucose transport, basal or insulin-stimulated, results in enhanced apoptosis at this stage which manifests later in pregnancy as a malformation or miscarriage. In preliminary studies, we have cloned a novel, insulin-regulated glucose transporter, GLUT8, which is expressed at high levels in the blastocyst stage in the mouse. Unlike the other glucose transporters, GLUT8 is localized intracellularly; however, upon insulin-treatment is translocated to the plasma membrane. This movement corresponds to an increase in glucose uptake. Also in preliminary studies, we have discovered that GLUT8 affects blastocyst survival. A decrease in GLUT8 expression by antisense oligonucleotides leads to an increase in apoptosis at the blastocyst stage as detected by TUNEL assay. These antisense embryos when transferred back to foster mice experience a higher rate of pregnancy failure and resorption compared to sense treated embryos. Thus, we hypothesize that the mammalian blastocyst represents the earliest insulin-regulated tissue and responds to insulin or IGF-l by translocation of GLUT8. We further postulate that establishment of this distinct glucose transport system is crucial for this stage of development. Dysregulation of this transport system may be responsible for increased apoptosis experienced under conditions of hyperinsulinemia as seen in polycystic ovary syndrome, and thus for the increased rate of pregnancy losses experienced by these women. The three aims to be pursued are: 1. Where is GLUT8 located intracellularly and in which cell type is GLUT8 expressed in the mammalian blastocyst? 2. How does IGF-1/insulin stimulate GLUT8 translocation and glucose uptake and how does this compare to GLUT4 translocation in skeletal muscle and adipocytes? 3. What is the physiological role of GLUT8 and how does decreased expression of this protein induce apoptosis at the blastocyst stage? Can triggering of GLUT8 translocation rescue the blastocyst from hyperinsulinemic conditions? Upon completion of the proposed research, we expect to have determined the location, mechanism and function of this novel glucose transporter expressed predominantly at the blastocyst stage. The role of this transporter in pathologic conditions and the ability to manipulate this transport system to improve embryo development and pregnancy outcome will be elucidated. Thus, these results are ultimately expected to have a significant impact on the field of reproductive biology.